We have modified our image averaging procedures (used previously to study the structure of dynein arms on outer microtubule doublets in Chlamydomonas axonemes) to study the central pair complex in this organism. Mitchell has recently characterized a Chlamydomonas flagellar mutant (cpc1) that lacks a central pair microtubule-associated complex. Cross-sections of wild-type, cpc1, and another central pair mutant, pf6, were imaged and negatives were sent to the Boulder 3-D laboratory. The negatives were digitized at a pixel size of 0.9 nm and central pair complexes in the images were extracted, self-aligned and then averaged. The resulting image averages showed dramatic structural defects in both mutant strains. The central pair microtubules in wild-type flagella have two projections off microtubule 1 (C1a and C1b) and two projections off microtubule 2 (C2a and C2b). The pf6 mutant strain was missing central pair projection C1a. In contrast, the cpc1 strain was missing the central pair projection C1b. Biochemical studies are currently underway to identify the proteins deficient in the mutant strains. This is the first study to apply image averaging techniques to study the fine structure of the central pair complex in Chlamydomonas. It should provide insight into the role of these structures in flagellar motility. C8